1. Field of the Invention
The present invention relates to somatostatin analogues and their use in diagnosis and therapy. The invention also relates to pharmaceutical compositions comprising the novel analogues.
2. Description of the Related Art
Somatostatin (somatotroph release-inhibiting factor), was initially discovered as a hypothalamic neurohormone that inhibits growth hormone secretion. It is a widely distributed peptide in both the central and peripheral nervous system and is also present in peripheral tissues including the endocrine pancreas, gut, thyroid, adrenals and kidneys. In addition, somatostatin is produced by inflammatory and immune cells as well as many cancer cells.
In mammals, two forms of bioactive peptides, somatostatin 14 and somatostatin 28 are found. They are produced by tissue-specific proteolytic processing of a common precursor. The natural somatostatin peptides have a short half-life, which is why many somatostatin analogues have been synthesized. Among them, octreotide, lanreotide and vapreotide have been intensively investigated and are in clinical use for the medical treatment of acromegaly and neuroendocrine tumors. These octapeptides retain the amino acid residues (or substitutes) within a cyclic peptide backbone that are involved in the biological effect of the peptide (Phe7 or Tyr7, D-Trp8, Lys9 and Thr10 or Val10) and display markedly increased stability.
The biological effects of somatostatin are mediated by specific plasma membrane receptors that have been identified in normal and neoplastic tissues by binding studies and receptor autoradiography techniques. Five somatostatin receptor genes have been cloned from human and mammalian libraries and designated sst1 to sst5 receptors. The sst subtypes belong to the family of G protein-coupled receptors with seven transmembrane-spanning domains and present a high degree of sequence identity (39–57%). The sequence differences reside in the extracellular and intracellular domains and are probably responsible for their signalling specificity.
All somatostatin receptors bind somatostatin 14 and somatostatin 28 with a high affinity (nM range), although with a slightly higher affinity for somatostatin 14. However, the receptors show major differences in their affinities for peptide analogues. Analogues that are known to date exhibit a low affinity for sst1 and sst4 whereas they bind the sst2 and sst5 receptor with a high affinity, comparable to that of somatostatin 14 and bind the sst3 receptor with moderate affinity.
In addition to its effect on secretion and intestinal motility, somatostatin inhibits the proliferation of normal as well as tumor cells. The antiproliferative action of somatostatin can be signalled via the five sst receptors which initiate pertussis toxin-sensitive G protein-dependent cell growth arrest or apoptosis according-to receptor subtypes and target cells.
When expressed in CHO cells, ligand-activated sst1, sst2A, sst4, and sst5 receptors inhibit mitogenic signal of serum or growth factors as a result of hypophosphorylation of the retinoblastoma gene product (Rb) and G1 cell cycle arrest.
However, distinct signal transduction pathways are involved in the somatostatin-induced G1 cell cycle arrest depending on receptor subtype. The sst1 receptor mediates cell growth arrest through the stimulation of the tyrosine phosphatase SHP-2, activation of the Ras/MAP kinase ERK pathway and induction of the cyclin-dependent kinase inhibitor p21waf1/Cip1, whereas the sst5 receptor acts by a mechanism involving a dephosphorylation cascade leading to inhibition of guanylate cyclase, cGMP-dependent protein kinase G and MAP kinase ERK 1/2.
The antiproliferative effect mediated by the sst2 receptor results from the activation of the tyrosine phosphatase SHP-1 and the dephosphorylation of activated growth factor receptors thus leading to the negative regulation of growth factor-induced mitogenic signalling.
In addition, somatostatin-activated SHP-1 induces a G1 cell cycle arrest, upregulates the cyclin-dependent kinase inhibitor p27Kip1 leading to the accumulation of hypophosphorylated Rb.
The antiproliferative effect of somatostatin can also result from apoptosis. Apoptosis is induced by sst3 as a result of the induction of p53 and Bax. In human pancreatic cancer cells expressing mutated p53 and devoid of endogenous sst2 receptor, correction of the deficit by expression of sst2 receptor induces an increase in cell death indicating that somatostatin can induce apoptosis by p53-dependent and p53-independent mechanisms.
The antiproliferative effects of somatostatin result from its actions via the endocrine pathway, but evidence exists that somatostatin can also act via an autocrine/paracrine pathway. Immunoreactive somatostatin has been found in somatostatin receptor-positive normal and tumor cell types such as endocrine, lymphoid cells, macrophages, breast cancer cells, colonic tumor cell and additionally, somatostatin mRNA is detected in a wide variety of neuroendocrine tumors known to express somatostatin receptors. Correction of the sst2 receptor deficit in human pancreatic cancer cells by sst2 receptor expression induces a negative autocrine loop in the absence of exogenous ligand, which is due to sst2 receptor-induced expression and secretion of endogenous sst2 ligand (somatostatin 14 and somatostatin 28). This results in inhibition of cancer cell proliferation and reversion of cell tumorigenicity in vitro and in vivo after xenografts in nude mice.
The somatostatin effect on tumor growth may be the result of indirect effects of the peptide resulting from the inhibition of secretion of growth-promoting hormones and growth factors which specifically regulate tumor growth. For example, the secretion of insulin-like growth factor-1 (IGF-1) which is produced by hepatocytes through GH-dependent and -independent mechanisms is negatively controlled by octreotide as a result of an effect on GH secretion and the sst2 and sst5 receptors have been demonstrated to be implicated in this effect. In addition, somatostatin can decrease IGF gene expression. Somatostatin also inhibits angiogenesis. Overexpression of peritumoral vascular somatostatin receptors with high affinity for somatostatin and octreotide has been reported in human peritumoral colorectal carcinomas, small cell lung carcinoma, breast cancer, renal cell carcinoma and malignant lymphoma. This expression appears to be independent of receptor expression in the tumor. It may reflect the presence of sst receptors in the venous smooth muscle cells as well as endothelial cells and may allow a vasoconstriction resulting in local hypoxia of the tumor or inhibition of endothelial cell growth and monocyte migration. Sst2, sst3 or sst5 receptors might be involved in these effects.
Although the biological role and cellular distribution of each receptor subtype are not yet completely understood it is clear that the development of analogues binding to all somatostatin receptor subtypes, so-called pansomatostatin, has high potential.
In one embodiment herein, it is thus an object of the present invention to provide such new somatostatin analogues that bind to all five somatostatin receptors and have a higher half-life (high metabolic stability) than somatostatin itself.